The clinical problem addressed in this proposed study is the need to prevent or reverse the process of inhibitor antibody formation in congenital and acquired hemophilia A patients. Although the number of affected patients is relatively small, development of inhibitor antibodies can result in life-threatening bleeding episodes, where the cost of treatment can exceed one million dollars per patient, per year. The experimental goals of this project are (1) to identify the predominant T-cell epitopes in human factor VIM(hFVIII), both in healthy individuals and in patients who have developed an allo- or auto-immune ("inhibitor") response to hFVIII; (2) to characterize the cytokine profiles of the stimulated T cells (3) to describe the evolution of these T-cell epitopes in inhibitor patients over time; and (4) to modify these epitopes in peptides, in recombinant hFVIII domains, and in full-length hFVIII molecules by introducing point mutations. Our central hypothesis is that modification of T-cell epitopes in FVIII will lead to development of a less immunogenic "next-generation" FVIII for use in cofactor replacement therapy. In addition, tolerance induction strategies with peptides or recombinant protein domains will be useful for patients who have already developed an inhibitor response to FVIII. Specifically, CD4+-enriched peripheral blood leukocytes will be isolated from hemophilia A patients as well as from healthy individuals, cultured, and stimulated with hFVIII, pools of synthetic peptides corresponding to the hFVIII domains, individual peptides, recombinant hFVIII domains, and proteolytic digests of hFVIII domains. Blood samples will be obtained from inhibitor patients at regular intervals and their T cells will be assayed. Immunodominant peptides, or possibly combinations of peptides, will be identified, their sequences will be modified, and the new peptides will be tested in the same manner. Promising mutations will then be introduced into the corresponding domains of a factor VIII protein, and the new proteins will be tested for both T-cell stimulation and for preservation of normal hFVIII function.